In the era of HAART, HIV-1 persists as integrated, latent proviruses in reservoir cells of infected individuals, as a major obstacle for cure. Tonsillr CD4+ T cells are a major HIV-1 oral reservoir. Understanding host factors regulating HIV-1 latency in these cells and identify cell-specific latency promoting genes (LPGs) will be useful to develop new latency reversing agents (LRAs) for elimination of HIV-1 oral reservoirs. Our earlier RNAi screens successfully identified LPGs that restrict HIV-1 transcription and facilitate its latency, including BRD4 and SUPT16H. In this proposal, we propose to further characterize these two LPGs in HIV-1 latently infected tonsillar CD4+ T cells (AIM 1 and 2). Their contribution to HIV-1 latency, particularly in oral reservoir cells, is largely unknown and worthy f further characterization. Using our established pipeline, we will also be interested to identify ne LPGs (AIM 3). We believe our proposed study is comprehensive and allows a systematic view how LPGs contribute to HIV-1 latency. AIM 1: We are one of the leading groups to identify BRD4 as a LPG, and we will further characterize BRD4 in HIV-1 latently infected tonsillar CD4+ T cells. We will first create a HIV-1 latency cell model using primary tonsillar CD4+ T cells. BRD4 will be depleted to determine how it affects HIV-1 latency/reactivation. Our preliminary results indicated that P-TEFb mediates BRD4's effect, so we will further determine the impact of BRD4 on P-TEFb activity in tonsillar CD4+ T cells. To test the feasibility to target BRD4 pharmacologically for purging latent HIV-1, we will evaluate a set of new promising BET inhibitors to revert HIV-1 latency. AIM 2: SUPT16H is our newly identified LPG, so we will study it in HIV-1 oral reservoir cells as well. We will determine whether depletion of SUPT16H in tonsillar CD4+ T cells may affect HIV-1 latency/reactivation. We will also use ChIP-seq approach to determine the genome-wide association of SUPT16H, which may provide clue how SUPT16H facilitates HIV-1 latency. Since SUPT16H is a nucleosome chaperone protein, we will determine whether such activity of SUPT16H may alter for HIV-1 transcription. Our preliminary results suggest that depletion of SUPT16H further enhances a HDAC inhibitor to revert HIV-1 latency, so we will further investigate this result by broadly testing a set of HDACIs. AIM 3: Our earlier RNAi screens indicate that unbiased functional genomic tools are capable of systemically identifying previously unappreciated host proteins modulating HIV-1 replication. These screens also suggest that there are other potential LPGs pending for discovery. In this aim, we will use the newly emerged CRISPR/Cas9 functional genomic approach to systematically identify novel LPGs. We will perform comprehensive bioinformatic and biostatistic analyses to filter primary screening results and prioritize a set of the most promising LPGs (10~15 genes) for further validations in tonsillar CD4+ T cells, including confirmation of LPG depletion and re-evaluation of HIV latency reverting phenotype. Our ultimate goal is to translate this understanding to pharmacologically target a handful of LPGs for eradicating HIV-1 oral reservoirs.